Recombinant yeast, construction method thereof and application of recombinant yeast in preparing tyrosol and derivatives

A technology for recombinant yeast and tyrosol, applied in the field of microbial genetic engineering, can solve the problems of incomplete research on metabolic pathways and the inability to achieve industrial production of tyrosol.

Active Publication Date: 2020-05-12
SHANDONG HENGLU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this technology can increase the yield of tyrosol in yeast, the yield of tyrosol still cannot meet the requirements of industrial production
The synthesis of tyrosol in yeast is limited by a variety of metabolic pathways, and the related metabolic pathways have not been fully studied. Therefore, how to realize the industrialization of tyrosol yeast fermentation is still a technical problem that needs to be solved urgently

Method used

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  • Recombinant yeast, construction method thereof and application of recombinant yeast in preparing tyrosol and derivatives
  • Recombinant yeast, construction method thereof and application of recombinant yeast in preparing tyrosol and derivatives
  • Recombinant yeast, construction method thereof and application of recombinant yeast in preparing tyrosol and derivatives

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Construction of Bafxpk and Bbfxpk expression cassettes

[0066] The amino acid sequences of SEQ ID NO.1 and SEQ ID NO.2 were codon-optimized according to the codon preference of the host Saccharomyces cerevisiae, and the optimized nucleotide sequences SEQ ID corresponding to SEQ ID NO.1 and SEQ ID NO.2 were obtained After NO.3 and SEQ ID NO.4, carry out gene synthesis. The primer pair Bafxpk-F / Bafxpk-R and Bbfxpk-F / Bbfxpk-R were selected to amplify the expression gene of the target gene fructose-6-phosphate phosphoketolase (Bafxpk The nucleotide sequence of the fragment is shown in SEQ ID NO.3, and the nucleotide sequence of the Bbfxpk fragment is shown in SEQ ID NO.4). Using the genome of Saccharomyces cerevisiae CICC1964 as a template, use the primer pair U-F / U-R and D-F / D-RPCR to amplify the DNA fragments of the upstream and downstream homology arms, and use the primer pair Ptpi1-F / Ptpi1-R and Tgpm1-F / Tgpm1-R The promoter tpi1 and terminator gpm1 fragments were amp...

Embodiment 2

[0080] Construction of Bafxpk and Bbfxpk heterologous expression strains, taking Saccharomyces cerevisiae as an example:

[0081] The tyrosol synthetic strain CICC1964 of Saccharomyces cerevisiae was transformed by PEG / LiAc method, the single clone was selected by adding G418 resistance in the medium, the genome was extracted, and the primers were used for PCR verification of Yzaw-F / Yzaw-R to obtain SC- bafxpk and SC-bbfxpk strains.

Embodiment 3

[0083] Fermentation of tyrosol-synthesizing microorganisms, taking Saccharomyces cerevisiae as an example:

[0084] Pick a single clone on the plate of tyrosol-producing strain CICC1964 and SC-bafxpk and SC-bbfxpk, inoculate it into 5mL YPD liquid medium, cultivate it at 30~32℃, 200rpm for 24h, transfer it to 50mL YPD liquid medium medium, initial seeding OD 600 After culturing at 0.2, 30°C, 200rpm for 12h, transfer to 100mL YPD liquid medium, initially inoculate OD 600 0.2, the medium contains carbon sources such as 2% glucose or 2% sucrose or 2% glucose and 1% tyrosine respectively, after 24 hours of cultivation, add 2% glucose or 2% sucrose or 2% glucose and 1% Tyrosine and other carbon sources were fermented for a total of 72 hours. The concentration of tyrosol in the fermentation broth was detected by the HPLC method reported in the literature (Satoh et al., Journal of Agricultural and Food Chemistry, 60, 979-984, 2012). The yields of tyrosol under different carbon sou...

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Abstract

The invention relates to a recombinant yeast, a construction method thereof and an application of the recombinant yeast in preparing tyrosol and derivatives. The recombinant yeast is obtained by introducing an exogenous fructose-6-phosphate phosphoketolase expression gene into a modified yeast cell, and the modified yeast cell is a yeast cell with a metabolic pathway of synthesizing tyrosol through 4-erythrose phosphate and phosphoenolpyruvic acid. The invention first discloses the fact that in a process of expressing fructose-6-phosphate phosphoketolase in yeast, fructose-6-phosphoric acid iscatalyzed into erythrose-4-phosphoric acid and acetyl phosphoric acid while 1,6-fructose diphosphate is synthesized, and xylulose-5-phosphoric acid is catalyzed into glyceraldehyde-3-phosphoric acidand acetyl phosphoric acid, so that distribution of the carbon metabolism flow in yeast is changed, synthesis of an important intermediate substance erythrose-4-phosphoric acid in tyrosol biosynthesisis enhanced, the metabolic pathway for synthesizing tyrosol is optimized, and the yield of tyrosol and derivatives such as hydroxytyrosol and the like is improved.

Description

technical field [0001] The present invention relates to recombinant yeast, its construction method and its application in the preparation of tyrosol and its derivatives, in particular to a strain through exogenous fructose-6-phosphate phosphoketolase (Fructose-6-phosphate phosphoketolase, fxp) The expression gene is introduced into yeast, and the method for efficiently producing tyrosol and its derivatives by using the strain belongs to the technical field of microbial genetic engineering. Background technique [0002] Tyrosol is a natural antioxidant derived from olive oil and a derivative of phenylethyl alcohol. Also known as salidroside aglycon, it is the main medicinal active ingredient of rhodiola rosea, and is the precursor of salidroside and hydroxytyrosol. It can protect cells from oxidative damage. It is a phenolic compound with important industrial value. Tyrosol and its derivatives are the synthetic precursors of various organic compounds. Tyrosol can be used in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P7/22C12R1/865C12R1/645
CPCC12N9/88C12P7/22C12Y401/02022C12N15/81C12R2001/865C12N15/70C12N15/815Y02E50/10C12N1/16C12N15/52C12N2510/02C12N2800/102C12Y103/01012C12Y401/01025C12Y504/99005
Inventor 方诩郭伟侯少莉
Owner SHANDONG HENGLU BIOTECH CO LTD
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